咪唑克生对脓毒症小鼠的器官保护及抗氧化应激分子机制研究

发布时间：2018-01-07 00:33

  本文关键词：咪唑克生对脓毒症小鼠的器官保护及抗氧化应激分子机制研究　出处：《重庆医科大学》2017年硕士论文　论文类型：学位论文

  更多相关文章： 咪唑克生 脓毒症 氧化应激 核因子E2相关因子2

【摘要】：目的:探讨咪唑克生(IDA)对脂多糖诱导脓毒症小鼠的器官保护作用及抗氧化应激分子机制。方法:(1)将60只成年C57BL/6小鼠随机分为对照组(12只,腹腔注射磷酸盐缓冲液)、LPS组(12只,腹腔注射LPS 10mg/kg)、低剂量组(12只,腹腔注射LPS 10mg/kg和IDA 1mg/kg)、中剂量组(12只,腹腔注射LPS 10mg/kg和IDA 2mg/kg)和高剂量组(12只,腹腔注射LPS10mg/kg和IDA 4mg/kg),于制模后24h处死所有小鼠,采集血和肝、肺、肾脏器组织标本。全自动生化分析仪检测血清丙氨酸氨基转移酶(ALT)和天冬氨酸氨基转移酶(AST)的水平;化学比色法检测肝组织匀浆中丙二醛(MDA)含量以及总超氧化物歧化酶(SOD)、过氧化氢酶(CAT)、总谷胱甘肽过氧化物酶(GPx)的活性;苏木精-伊红染色观察肝、肺、肾组织病理学改变;Western Blot法检测肝组织匀浆中HO-1、NQO-1、Nrf2蛋白表达;免疫组化法检测肝、肾组织Nrf2的表达。(2)体外培养小鼠巨噬细胞系RAW264.7细胞,随机分为四组:对照组、LPS组、治疗组及IDA组,对照组仅用完全培养基培养,LPS组给予10μg/ml LPS刺激,LPS+IDA组同时给予10μg/ml LPS+100μM IDA作用,IDA组给予100μM IDA处理。以2,7-二氢二氯荧光素二乙酸酯(DCFH-DA)为荧光探针,运用流式细胞仪及荧光显微镜检测细胞内活性氧(ROS)水平;Western Blot法检测细胞中HO-1、NQO-1、Nrf2蛋白表达,细胞免疫荧光检测细胞内HO-1的表达。结果:(1)LPS刺激24h,LPS组小鼠血清中ALT、AST水平均显著高于对照组[ALT(U/L):88.10±12.05比35.93±3.02;AST(U/L):311.97±75.04比136.77±10.80;均P0.01]。IDA能有效降低LPS所致的小鼠血清转氨酶的异常升高[ALT(U/L):低剂量组68.40±5.17比88.10±12.05(P=0.06),中剂量组51.10±5.66比88.10±12.05(P0.01),高剂量组44.27±9.10比88.10±12.05(P�d0.01);AST(U/L):低剂量组257.73±51.97比311.97±75.04(P=0.36),中剂量组194.37±20.84比311.97±75.04(P=0.06),高剂量组161.13±27.91比311.97±75.04(P�d0.05)]。肝组织匀浆中,LPS组的MDA水平显著高于对照组[(6.37±1.45)nmol/mg比(1.13±0.25)nmol/mg,P0.01],抗氧化蛋白酶(SOD、CAT及GPx)的活性显著降低[SOD(U/mg):0.97±0.35比3.83±0.71;CAT(U/mg):30.67±7.02比70.33±8.50;GPx(U/mg):8.33±2.52比30.33±5.51,;均P0.01]。然而,咪唑克生治疗则可呈剂量依赖性地减少丙二醛(MDA)水平[低剂量组(4.83±0.80)nmol/mg比(6.37±1.45)nmol/mg(P=0.18),中剂量组(3.20±0.62)nmol/mg比(6.37±1.45)nmol/mg(P0.05),高剂量组(2.30±0.46)nmol/mg比(6.37±1.45)nmol/mg(P�d0.01)]及改善抗氧化蛋白酶(SOD、CAT及GPx)活性的抑制[SOD(U/mg):低剂量组1.47±0.31比0.97±0.35(P=0.14),中剂量组2.36±0.47比0.97±0.35(P0.05),高剂量组3.27±0.70比0.97±0.35(P�d0.01);CAT(U/mg):低剂量组50.33±13.50比30.67±7.02(P=0.09),中剂量组67.67±13.50比30.67±7.02(P�d0.05),高剂量组80.67±13.01比30.67±7.02(P�d0.01);GPx(U/mg):低剂量组15.67±4.04比8.33±2.52(P=0.06),中剂量组21.33±4.51比8.33±2.52(P�d0.05),高剂量组35.33±9.07比8.33±2.52(P�d0.01)]。IDA还可明显改善小鼠肝、肺、肾的病理改变,并且可增加肝组织中HO-1、NQO-1及Nrf2及肾组织中Nrf2的表达。(2)体外实验中,与对照组相比,RAW264.7细胞受到LPS刺激后,细胞内ROS明显增加(P�d0.01),而IDA能显著降低LPS诱导的ROS异常升高(P�d0.05)。同时,与LPS组相比,LPS+IDA组中HO-1、NQO-1及Nrf2的蛋白表达明显增加。结论:IDA通过激活巨噬细胞Nrf2信号通路发挥抗氧化应激作用,从而减轻小鼠脓毒症时的多器官损害。
[Abstract]:Objective: To investigate the effects of idazoxan (IDA) on lipopolysaccharide induced septic mouse organ protective effect and antioxidative molecular mechanism. Methods: (1) a total of 60 adult C57BL/6 mice were randomly divided into control group (12 rats, intraperitoneal injection of phosphate buffer), LPS group (12 rats, intraperitoneal injection of LPS 10mg/kg). The low dose group (12 rats, intraperitoneal injection of LPS 10mg/kg and IDA 1mg/kg), middle dose group (12 rats, intraperitoneal injection of LPS 10mg/kg and IDA 2mg/kg) and high dose group (12 rats, intraperitoneal injection of LPS10mg/kg and IDA 4mg/kg), in the system of 24h after all the mice were sacrificed, blood and liver, lung and kidney for tissue samples. The serum alanine amino transferase automatic biochemical analyzer (ALT) and aspartate aminotransferase (AST) levels were measured; liver tissue homogenate colorimetric method (MDA) content and superoxide dismutase (SOD), catalase (CAT), total glutathione. Oxidation Peroxidase (GPx) activity; hematoxylin eosin staining of liver, lung, renal pathological change; HO-1, detection of Western in liver tissue homogenate in Blot NQO-1, Nrf2 protein expression; immunohistochemistry of liver, expression of Nrf2 in renal tissue. (2) mouse macrophage RAW264.7 cells cultured in vitro. Were randomly divided into four groups: control group, LPS group, IDA group and treatment group, the control group only with complete medium, LPS group was given 10 g/ml LPS stimulation, while group LPS+IDA was treated with 10 g/ml LPS+100 M IDA, IDA group was given 100 M IDA to two 2,7-. Two hydrogen chloride two fluorescein acetate (DCFH-DA) as a fluorescence probe by flow cytometry and fluorescence microscopy to detect intracellular reactive oxygen species (ROS); HO-1, Western Blot in NQO-1 cells, the expression of Nrf2 protein expression, cell immunofluorescence detection of HO-1 in cells. Results: (1) LPS stimulated 24h, serum LPS group mice in ALT, AST 姘村钩鍧囨樉钁楅珮浜庡�圭収缁刐ALT(U/L):88.10卤12.05姣，

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